SUMMARY: Production of infectious HIV-1 particles requires a coordinated intracellular process in which Gag proteins assemble into immature capsids while simultaneously encapsidating the unspliced HIV-1 RNA that becomes the packaged HIV-1 genome. Exciting advances in the field have led to identification of novel features of unspliced HIV-1 RNA that are characteristic of released virus. However, to date, the identity of the complexes in which assembly and packaging occur within infected cells remains controversial and poorly understood. The current proposal addresses the following important questions: Which of the intracellular complexes containing Gag and unspliced HIV-1 RNA have features characteristic of packaging? What is the proteome of complexes containing Gag and unspliced HIV-1 RNA, and which of the proteins in these complexes are functional? Can small molecules that inhibit these complexes be identified and used as tools for understanding of complexes containing Gag and unspliced HIV-1 RNA? Studies in Aim 1 propose to systematically analyze lysates of infected T cells to identify the spectrum of intracellular complexes that display three key features found in release virus. Intracellular complexes that display one or more of these features are likely to be packaging intermediates. Aim 2 seeks to define the proteome of intracellular complexes containing Gag and unspliced HIV-1 RNA, including those that display key features of released virus. This aim will also identify functional proteins in these complexes, and examine how these proteomes can be modulated. Finally, in Aim 3, preliminary data is provided in support of a potent small molecule - PAV117 - that appears to inhibit HIV-1 virus production by acting during Gag assembly. Since none of the inhibitors of HIV-1 late events that are currently under investigation block virion production, PAV117 appears to be a first-in-class inhibitor of immature capsid assembly. Studies proposed in Aim 3 will utilize PAV117 to better understand intracellular events in HIV-1 assembly. The intracellular target of PAV117 in infected T cells will be identified using analogs of PAV117 that are biotinylated or modified for click chemistry. Additionally, a newly isolated PAV117-resistant virus will be studied to determine how it overcomes inhibition by PAV117. Finally, the proviral mutations responsible for PAV117 resistance will be identified. Together these studies hold great promise for advancing our understanding of the sequence of events that leads to assembly and packaging, definitively identifying the intracellular complexes in which these events occur, and gaining insights into the mechanism underlying a novel inhibitor of immature capsid assembly.